Methods for detecting and localizing DNA mutations by microarray

ABSTRACT

This disclosure provides methods for detecting and localizing DNA mutations by DNA microarray. In various embodiments, the described methods include use of restriction endonuclease(s) and/or mismatch-recognition nuclease(s) to detect and/or localize mutations. In one representative method, reference and target DNA are digested using one or more restriction endonucleases, resultant DNA strands are labeled (e.g., using a DNA polymerase), and the labeled mixture of DNAs is hybridized to a microarray. In another representative method, reference and target DNA are denatured and annealed to form a mixture containing heteroduplex DNA, one or more mismatch-recognition nuclease(s) are used to nick or cleave at least a portion of the heteroduplex DNA, resultant DNA strands are labeled (e.g., using a DNA polymerase) and the labeled mixture of DNAs is hybridized to a microarray.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 10/236,598, filed Sep. 6,2002, now U.S. Pat. No. 7,141,371, issued Nov. 28, 2006, the entiredisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Detection and identification of simple nucleotide mutations and/orpolymorphisms among individuals are very important in many biologicalfields, ranging from biomedical research in hereditary diseases toecology and evolutionary biology. Identification of the location ofmutations involved in heritable diseases can provide clues for thediagnosis, prognosis and therapeutic treatment of such diseases.However, it is very difficult to localize most of the mutations into avery small region (several kilo-base pairs) or a single gene at agenomic level.

Sequencing of the genome of different individuals is the moststraightforward method for mutation detection, but it is expensive andtime-consuming. Some methods have been developed to detect mutationswithout direct sequencing. Linkage and association mapping usepolymorphic markers to approximate the chromosomal location of themutations. This method is very efficient to localize a mutation into aquite large genomic region (several hundreds kilo-base pairs), but veryslow to further localize it into a single gene or a small region(several kilo-base pairs). Restriction fragment length polymorphism(RFLP) is good for mutation detection and sequence comparison but cannot efficiently define the location of a mutation either if wholegenomic DNA is used as the starting material.

Most of the currently developed methods or techniques are suitable fortesting mutations in a small region or a handful of genes, but are notgood (if not impossible) for localizing mutations or polymorphisms at agenomic level. For example, in polynucleotide microarray hybridizationmethods (e.g. U.S. Pat. No. 5,837,832 (Chee et al.), and U.S. Pat. No.6,376,191 (Yu et al.)) mutations in every single base position aredetected by a set of four primers. To fully detect a 10 kb region, thesemethods will need about 10,000 sets of primers to be spotted on amicroarray slide. Single stranded conformational polymorphism (SSCP) anddenaturing gradient gel electrophoresis (DGGE) (Orita et al (1989) PNAS86:2766; Myers et al (1985) N. A. R. 13:3131; White et al (1992)Genomics 5:301; Mills et al (1994) Biochem. 33:1797) methods are basedon the observations that DNA sequence variations can cause DNAelectrophoretic mobility changes. These two methods only work with shortDNA molecules and can not screen many genes simultaneously.

The two methods I present in this invention have great potential todetect and localize DNA mutations at a genomic level simultaneously andrapidly. The method using restriction endonuclease(s) to detectmutations is termed RE microarray method. The other method usingmismatch-recognition endonuclease(s) is named MR microarray method. Theprinciple of the methods is illustrated in FIG. 1.

BRIEF SUMMARY OF THE INVENTION

Mutation detection is very important for the diagnosis, prognosis andtherapeutic treatment of heritable diseases. However, none of the knownmethods can efficiently detect and define the location of mutations at agenomic level. In this invention, two methods are provided for such apurpose. The RE mutation detection microarray method used restrictionendonuclease to detect mutations and the MR microarray method usedmismatch-recognition nuclease to detect mutations. In the RE microarraymethod, the reference and target DNAs were completely digested with arestriction endonuclease. If a mutation caused the elimination of arestriction site of the endonuclease, the two restriction fragments inthe reference DNA flanking the position of the mutation would in thetarget DNA become one large fragment spanning the position of themutation. After denaturation and annealing, one single strand from theabove two fragments of the reference DNA could anneal with one strand ofthe above large fragment of the target DNA to form a partiallydouble-stranded DNA and DNA polymerase could then use the short strandof this DNA as a primer and the long strand as a template to label theshort strand DNA by incorporating fluorescent nucleotides into newlysynthesized DNA. Therefore, by this mechanism only the DNA strandsflanking the mutation could be labeled. When hybridized to a microarrayslide, the labeled DNA would bind to the spot whose DNA has the samesequence as the labeled DNA. By identification of the sequence of thespot DNA, the mutation would then be localized at or around this DNAsequence region, which can be as small as several kilo-bases. Similarly,a mutation that created a restriction site of the applied restrictionendonuclease can also be detected and localized by this method.

In the MR mutation detection microarray method, reference DNA and targetDNA were mixed, fragmented (by a restriction endonuclease), denaturedand annealed to form heteroduplex DNA (between a single strand of areference DNA fragment and a strand of the corresponding target DNAfragment carrying a mutation) as well as homoduplex DNA. Theheteroduplex DNA was then specifically recognized and cleaved around themismatch site into two short fragments by a mismatch-recognitionnuclease while its corresponding homoduplex DNA and other homoduplex DNAwould not be cleaved and kept full length. After re-denaturation andre-annealing of the nuclease-treated DNA mixture, a single strand of thecleaved short DNA fragments could anneal to a single strand of itscorresponding full length DNA fragment to form a partiallydouble-stranded DNA. As described in the RE microarray method, DNApolymerase could then use this partially double-stranded DNA to labelthe DNA strands flanking the mutation and after hybridization to a DNAmicroarray the location of the mutation would be identified.

In practice, both methods also used a differently labeled control DNA.The control DNA is the reference DNA alone or the target DNA alonetreated in the same way as the mixture of the reference and target DNAas described above but labeled differently from the mixture of referenceand target DNA. The differently labeled control DNA would then becombined with the labeled mixture DNA and hybridized to a microarrayslide. The mutations would be detected and localized by identifying thesequence of each DNA whose microarray spot had a higher ratio of thelabel signal from the mixture DNA to the label signal from the controlDNA. Such a control could reduce the effect of non-specific cutting ordigestion by the nuclease and varied DNA amounts of different microarrayspots, etc.

To prove the practicability of the methods, mutations in plasmids weredetected and localized. A wild type plasmid was divided into tworegions, L and R, and two DNA molecules separately from the two regionswere used to spot onto slides to make mini microaffays. Three mutantplasmids (target DNAs) were compared with the wild type parental plasmid(reference DNA) by the methods. One of the mutant plasmids had amutation that caused the elimination of an Ase I restriction site andthe RE mutation detection microarray method successfully detected andlocalized the mutation into the L region. The other two mutant plasmidseach had a single nucleotide mutation and the MR mutation detectionmicroaffay method correctly detected and identified the location of themutations. These experimental results indicate that the methods arepractical and successful in mutation detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the principal of the method.

FIG. 2 shows the map of the parental plasmid DNA. The locations of themutations in target plasmid are also indicated.

FIG. 3 a shows the difference of restriction patterns between a wildtype plasmid and a mutant plasmid where an Ase I site is missing.

FIG. 3 b shows that the mutation that caused the missing of an Ase Isite in a mutant plasmid can be detected and correctly localized by theRE microarray method.

FIG. 4 shows the cleavage of a heteroduplex DNA by Cel I nuclease.

DETAILED DESCRIPTION OF THE INVENTION

Materials used in this invention are listed as follows:

All the restriction endonucleases used were from New England BiolabsInc. Taq DNA polymerase, its reaction buffer and dNTPs were from RocheApplied Science. QIAquick gel extraction kit was used for isolating DNAfragments from agarose gel and QIAquick PCR purification kit was used topurify DNA fragments from enzymes, nucleotides and salts (from QiagenGmbH). Cy3 and Cy5-labeled dCTPs were from Amersham Biosciences.

Wild type (reference DNA) and mutant plasmids (target DNAs) used in thiswork were from Tom H. Stevens lab at the University of Oregon. pKH28 wasconstructed by inserting a 795bp fragment carrying a wild type VMA21gene in between the Sac I and Kpn I sites of pRS 316. pLG119 has a T toA substitution at 2106bp. pLG120 has an A to C substitution at 2109bpand pLG 125 has an adjacent two nucleotides substitution (AT to GC) at2126bp and 2127bp, which happens to eliminate an Ase I cutting site(ATTAAT) by changing the first AT to GC. In this work pKH28 is calledwild type plasmid DNA or reference DNA. pLG119, 120 and 125 are calledmutant plasmid DNA or target DNA.

The DNA microarray slides were made according to the protocols publishedat web site http://www.microarrays.org, which is maintained by StanfordUniversity. Plasmid pKH28 and its derivatives pLG119, 120 and 125 allhave two Sca I restriction sites (FIG. 2). In this invention, the twoSca I sites were used to separate the plasmid into two regions (L and S)(see FIG. 2) and the novel methods were tested to see if it couldlocalize the mutations into a correct region. Therefore, a very simpleDNA microarray was made, which only contains two different DNAmolecules: a large Sca I fragment molecule (L region DNA) and a smallSca I fragment molecule (S region DNA). These two DNA fragments wereisolated from plasmid pKH28 DNA after digestion with Sca I, separationon agarose gel and purification. The two DNAs were spotted onto glassslides in a tandem repeat order as L, S for six times. Therefore, thereare six spots for each DNA per microarray slide.

Cel I nuclease, a mismatch-recognition enzyme, was prepared from celeryaccording to a published method (Yang et al (2000) Biochem. 39:3533)with some modifications and less steps of purification. Briefly, 10 kgof fresh celery stalks was homogenized with a juicer and the juice wascollected and adjusted to the composition of buffer A (100 mM Tris-HCI,pH7.7, 0.5 M KCL and 100 μM PMSF). The suspension was centrifuged at8000 g for 30 min and the supernatant was collected. 10 ml ofConcanavalin A-sepharose (from Sigma) resin was added into the clearedsupernatant and gently shaken overnight at 4° C. The resin was thenpelleted and washed twice with buffer A by two cycles of centrifugation(at 3000 g for 30 min) and resuspended. Cel I was then eluted from theresin with 80 ml of buffer A containing 0.3 M α-methyl-mannoside byincubation at 4° C. for four hours with gentle shaking. Aftercentrifugation, the Cel I nuclease solution (supernatant) was collected,aliquot and stored at −20° C. When needed, an aliquot of the enzymewould be thawed and used. For the cleavage of mismatched double-strandedDNA, the best condition for a reaction of 2 μg DNA in a 30 μl finalvolume in 1X Cel I reaction buffer (20 mM Tris-HCl pH7.4, 25 mM KCL, 10mM MgCl₂ was 1 μl of this Cel I preparation and 30 min incubation at 37°C.

The following examples are intended to illustrate the present inventionand should in no way be construed as limiting the invention.

EXAMPLE 1

Detection and Localization of DNA Mutations by the RE Mutation DetectionMicroarray Method

Restriction fragment length polymorphism (RFLP) is one of the mostpopularly used methods for mutation detection and sequence comparison.However, it can not define the location of the mutation. In thisexperiment, a microarray method is developed for rapid detection andlocalization of DNA mutations.

A mutation in a mutant plasmid pLG125 caused the missing of an Ase Isite, which is present in the wild type plasmid pKH28 (see Materials andFIG. 2).

One microgram (μg) each of pLG125 and pKH28 was double digested with PvuII and Ase I in NEB buffer 2 plus BSA, purified with QlAquick PCRpurification kit and resuspended in 30 microliters (μl) of EB buffer (10mM Tris-HCI, pH8.0). 5 μl of the above digested pKH28 DNA was mixed with5 μl of the above pLG125 DNA to give a hetero-mixture DNA. 10 μl of theabove digested pKH28 was also taken and called a control DNA. To labelthe DNA, the following chemicals were added to each of the mixtures: 2μl of Taq DNA polymerase buffer (with 2.0 mM MgSO₄,5 μl of dATP, dTTPand dGTP mixture each at a concentration of 2 mM, 1 μl of 2 mM dCTP, 1μl of 1mM Cy5-dCTP (for hetero-mixture DNA) or Cy3-dCTP (for the controlDNA), and 1 μof Taq DNA polymerase (1 unit). A PCR machine was then usedto complete the labeling reaction by a program that the first step is95° C. 2 min to denature double-stranded DNA, then run 15 cycles of 95°C. 1 min and 72° C. 2 min, followed by a step of 72° C. 15 min andfinally stop the reaction by quickly cooling to 4° C. The labeledreaction mixtures were combined, purified by QIAquick PCR purificationkit and eluted with 30 μl of EB buffer (10 mM Tris-HCl, pH8.0). 10.5 μlwas taken out to mix with 3 μl of 20X SSC, 1.1 μl 20 mg/ml polyA and0.42 μl of 10% SDS. After being heated at 100° C. for 2 min and brieflycentrifuged, the mixture was then loaded onto a microarray slide forhybridization at 65° C. for 4-16 hours. After scanning of the microarrayslides, it was found that in the same microarray slide the Cy5/Cy3 ratioof any spot of the L region DNA was about 5 to 9 times higher than thatof any spot of the S region DNA (see FIG. 3). This clearly localizes themutation in the L region in pLG125 plasmid, which is correct, indicatingthis method is feasible in mutation detection and localization.

EXAMPLE 2

Detection and Localization of DNA Mutations by the MR Mutation DetectionMicroarray Method

This experiment comprises four major steps: formation of heteroduplexand homoduplex DNA; treatment of the duplexes with amismatch-recognition endonuclease to cleave the heteroduplexes at themismatch site; labeling the cleaved DNA by DNA polymerase-mediatedincorporation of modified nucleotides; and hybridizing the labeled DNAto DNA microarray.

There are a few mismatch-recognition endonucleases such as SP nucleaseand Mung Ben nuclease. Here, Cel I nuclease was chosen because of itshigh specificity of cleavage at all single nucleotide substitutions(Oleykowski et al (1998) Nucleic Acids Research 26: 4597).

To detect and localize a mutation in plasmid pLG119, both pLG119 andwild-type plasmid pKH28 were digested with Sca I and then purified withQIAquick PCR purification kit. One microgram of this Sca I-digestedpLG119 (pLG119/Sca I) was mixed with one microgram of the Sca I-digestedpKH28 (pKH28/Sca I) in 30 microliters of 2X Cel I reaction buffer. As acontrol, two micrograms of the pKH28/Sca I were also resuspended in 30μl of 2X Cel I reaction buffer and would experience exactly the sametreatment as the wild type and mutant DNA mixture. The mixtures werethen denatured at 95° C. for 5 min, cooled to 85° C. quickly and thenslowly cooled down to 30° C. at a speed of 3° C. per hour. Thisdenaturation and annealing step was processed in a PCR machine. In thepKH28/Sca I and pLG119/Sca I mixture, both homoduplexes andheteroduplexes would form randomly. The pKH28/Sca I control would bemainly homoduplexes.

After the formation of heteroduplexes, 28 μl of sterile distilled waterwas added to each of the above DNA mixtures to get a total of 58 μl. Tocleave heteroduplex DNA, 1 μl of Cel I preparation was added into 29 μlof each of the mixtures and incubated at 37° C. for 30 min. Afterimmediate purification with a QIAquick PCR purification kit, each of theCel I-treated DNA mixtures was resuspended in 30 μl of LB buffer (10 mMTris-HCl, pH8.0). 6 μl of each was taken and loaded onto 1.0% agarosefor gel electrophoresis. It was shown that Cel I could cleaveheteroduplexes and produce two new smaller fragments (FIG. 4). It wasalso shown that Cel I had quite strong non-specific cutting becausethere was some smear on both of the lanes loaded with Cel I-treated DNA.This is consistent with a former publication (Sokurenko et al (2001)Nucleic Acids Research 29: e111), where Cel I caused significantnon-specific digestion of duplex DNA.

To detect the cleaved DNA, Taq DNA polymerase was used to addfluorescent Cy3 or Cy5-dCTP onto it. For this purpose, 10 μl of each ofthe above purified Cel I-treated DNA was mixed with 2 μl of Taq DNApolymerase buffer (with 2.0 mM MgSO₄,5 μl of dATP, dTTP and dGTPmixtures each at a concentration of 2 mM, 1 μl of 2 mM dCTP, 1 μl of 1mM Cy5-dCTP (for DNA from the mixture of pKH28/Sca I and pLG119/Sca I)or Cy3-dCTP (for the control DNA), and 1 μl of Taq DNA polymerase (1unit). A PCR machine was then used to complete the labeling reaction bya program that the first step is 95° C. 2 min to denaturedouble-stranded DNA, then run 15 cycles of 95° C. 1 min and 72° C. 2min, followed by a step of 72° C. 15 min and finally stop the reactionby quickly cooling to 4° C. The labeled reaction mixtures were combined,purified by QIAquick PCR purification kit and eluted with 30 μl of LBbuffer (10 mM Tris-HCl, pH8.0). 10.5 μl was taken out to mix with 3 μlof 20X SSC, 1.1 μl 20 mg/ml polyA and 0.42 μl of 10% SDS. After beingheated at 100° C. for 2 min and briefly centrifuged, the mixture wasthen loaded onto a microaffay slide for hybridization at 65° C. for 4-16hours.

The localization of the mutation can be achieved by comparing the ratios(Cy5/Cy3) of different DNA spots. With more than six repeats of theabove experiments and each experiment with 6 repeated spots for each DNAfragment, it was found that in the same microarray slide the Cy5/Cy3ratio of any spot of the L region DNA was 0.4-0.7 times higher than thatof any spot of the S region DNA. This predicts that the mutation caffiedby plasmid pLG119 was located on the large Sea I fragment, which iscorrect according to the known fact that the mutation is there. Thisindicates that though there is quite strong non-specific cutting by CelI nuclease, this method was still able to detect and localize the DNAmutation.

Plasmid pLG120, which has an A to C substitution, was also used to testthis method. A positive result very similar to that from pLG119 wasobtained again. This further proved the practicability of this methodand indicated the result of this method is repeatable and reliable. Thismethod should work on genomic microarray in a similar way and then DNAmutations or polymorphisms in individual genomes would be able to bedetected and localized rapidly.

1. A method comprising: a) digesting reference and target DNA using arestriction endonuclease, wherein the restriction endonuclease digeststhe reference and the target DNA into different DNA fragments around oneor more mutations that eliminates or creates a restriction site of theendonuclease, and mixing the reference and target DNA, thereby producinga digested mixture of reference and target DNA; b) labeling DNA strandsof the digested mixture of reference and target DNA flanking the one ormore mutations by one or more cycles of denaturation, annealing andlabeling with a DNA polymerase, wherein denaturation and annealingallows annealing of the different DNA fragments around one or moremutations from reference and target DNA to form a partiallydouble-stranded DNA duplex, and wherein the DNA polymerase labels theDNA of the partially double-stranded duplex by incorporating modifiednucleotides into newly synthesized DNA, thereby producing a labeledmixture of reference and target DNA; c) hybridizing a combination of thelabeled mixture of reference and target DNA and a labeled control DNA toat least one DNA array, which array comprises spots comprising at leastone DNA probe, at least one of the spots comprising a DNA probe from thetarget DNA or the reference DNA or both, and wherein the labeled controlDNA is (i) reference DNA alone or (ii) target DNA alone, treatedsubstantially as in a) and b) but labeled with a distinguishablemodified nucleotide; and (d) identifying the sequence of DNA of at leastone spot of the DNA array that has significantly higher ratio of labelsignal from the mixture of reference and target DNA compared to labelsignal from the control DNA, thereby detecting and localizing one ormore mutations in the target DNA as compared to the reference DNA. 2.The method of claim 1, wherein the restriction endonuclease is at leastone restriction endonuclease or an agent that binds to specific DNAsequence and cleaves or nicks DNA at or near the specific sequence, or acombination of two or more thereof.
 3. The method of claim 1, whereinthe DNA polymerase is a DNA polymerase or a mixture of two or moredifferent DNA polymerases.
 4. The method of claim 1, wherein spots ofthe DNA array comprise DNA fragments covering a whole genome.
 5. Amethod comprising: a) forming a mixture of heteroduplex and homoduplexDNA by denaturing and annealing a mixture of reference and target DNA toform heteroduplex DNA, homoduplex reference DNA, and homoduplex targetDNA; b) treating the mixture of heteroduplex and homoduplex DNA with amismatch-recognition molecule, thereby nicking or cleaving theheteroduplex DNA at or around a mismatch site; c) labeling DNA strandsby one or more cycles of denaturation, annealing and labeling with a DNApolymerase, wherein denaturation and annealing allows one short strandfrom the nicked or cleaved heteroduplex DNA to anneal with one longstrand from homoduplex DNA spanning the same mismatch site to form apartially double-stranded DNA duplex, and wherein the DNA polymeraseuses the short strand as a primer and the long strand as a template tolabel the short strand by incorporating modified nucleotides into newlysynthesized DNA, thereby producing a labeled mixture of reference andtarget DNA; d) hybridizing a combination of the labeled mixture ofreference and target DNA and a labeled control DNA to at least one DNAarray, which array comprises spots comprising at least one DNA probe, atleast one of the spots comprising a DNA probe from the target DNA or thereference DNA or both, and wherein the labeled control DNA is (i)reference DNA alone or (ii) target DNA alone, treated substantially asin a) and b) but labeled with a distinguishable modified nucleotide; ande) identifying the sequence of DNA of at least one spot of the DNA arraythat has a significantly higher ratio of label signal from the mixtureof reference and target DNA compared to label signal from the controlDNA, thereby detecting and localizing one or more mutations in thetarget DNA as compared to the reference DNA.
 6. The method of claim 5,wherein the mismatch-recognition nuclease is at least one nuclease thatcleaves or nicks heteroduplex DNA, or a mixture of different nucleases.7. The method of claim 5, wherein the mismatch-recognition nuclease is anaturally existing nuclease, an artificial enzyme constructed by fusinga mismatch-recognition protein or peptide to a nuclease, or acombination thereof.
 8. The method of claim 5, wherein the DNApolymerase is a DNA polymerase or a mixture of two or more different DNApolymerases.
 9. The method of claim 5, wherein spots of the DNA arraycomprise DNA fragments covering a whole genome.
 10. The method of claim1, wherein the modified nucleotides are labeled nucleotides thatcomprise a label that is fluorescent, enzymatic, chemiluminescent orradioactive, or nucleotides conjugated to a molecule that binds to alabel which is fluorescent, enzymatic, chemiluminescent or radioactive.11. The method of claim 5, wherein the modified nucleotides are labelednucleotides that comprise a label that is fluorescent, enzymatic,chemiluminescent or radioactive, or nucleotides conjugated to a moleculethat binds to a label which is fluorescent, enzymatic, chemiluminescentor radioactive.
 12. The method of claim 1, wherein c) further comprisesreacting the newly synthesized DNA with an agent that labels themodified nucleotides.
 13. The method of claim 5, wherein d) furthercomprises reacting the newly synthesized DNA with an agent that labelsthe modified nucleotides.
 14. The method of claim 5, wherein the mixtureof reference and target DNA is a mixture of fragmented reference andtarget DNA obtained by using at least one restriction endonuclease todigest the reference and target DNA.